PF-05231023 reduces lipid deposition in apolipoprotein E-deficient mice by inhibiting the expression of lipid synthesis genes

Fibroblast growth factor 21 (FGF21) is a peptide hormone that is primarily expressed and secreted by the liver. The hormone is crucial for regulation of glucose homeostasis, lipid metabolism, and energy balance. Compared with natural FGF21, FGF21 analogs have become drug candidates for the treatment of cardiovascular and metabolic diseases owing to their long half-life and greater stability in vitro. Apolipoprotein E (Apoe)-knockout (Apoe−/−) mice exhibit progressive disruptions in lipid metabolism in vivo and develop further atherosclerosis pathological features owing to Apoe deletion. Therefore, this study used an Apoe−/− mouse model to investigate the effects of a long-acting FGF21 analog (PF-05231023) on lipid metabolism and related parameters. Eighteen Apoe−/− female mice were fed a Western diet equivalent for 12 weeks, and then randomly assigned to intraperitoneally receive either physiological saline (the control group) or 10 mg/kg PF-05231023 (the treatment group) three times a week for seven consecutive weeks. Body composition, glucose tolerance, blood and liver cholesterol, triglyceride levels, liver vacuolization levels, peri-ovarian white adipocyte hypertrophy, aortic atherosclerotic plaque formation, and the expression of genes related to lipid metabolism in adipose tissue were subsequently assessed before and after treatment. The aortic atherosclerotic plaque area was reduced in mice in the PF-05231023 treatment group compared with that in the saline group. Although the effect of PF-05231023 on the plasma biochemical indexes of mice was small, it significantly reduced lipid levels and lipid droplet accumulation in the liver, and reduced adipocyte hypertrophy in white adipose tissue. Transcriptome analysis of adipose tissue showed that PF-05231023 treatment downregulated the expression of lipid synthesis-related genes and inhibited the sterol regulatory element binding transcription factor 1 gene, thereby improving lipid deposition. PF-05231023 effectively improved the lipid metabolism of Apoe−/− mice, demonstrating an anti-atherosclerotic effect and providing a scientific basis and experimental foundation for the clinical treatment of cardiovascular diseases by using long-acting FGF21 analogs.


Introduction
Excessive consumption of calorie-rich foods leading to obesityrelated diseases is now recognized as a major cause of disability around the world (1).Mechanistically, caloric intake that exceeds adipose tissue storage capacity is associated with the accumulation of ectopic lipids in non-adipose organs and the induction of low-grade tissue inflammation, endoplasmic reticulum stress, and insulin resistance (2,3).These metabolic defects increase the risk of serious diseases, including type 2 diabetes, non-alcoholic steatohepatitis, cardiovascular disease and various forms of cancer.However, to date, specific drugs for these diseases remain limited.
Fibroblast growth factor 21 (FGF21) is a peptide hormone that is primarily expressed and secreted by the liver and adipose tissue (4,5).The hormone acts in an endocrine manner and targets liver and adipose tissue (6).Increasing evidence suggests an important role for FGF 21 in the regulation of glucose and lipid homeostasis through multifaceted and inter-organ crosstalk.Moreover, FGF 21 has been recognized as a potential target for metabolic abnormalities (7).Although the role of FGF21 in obesity, diabetes, and non-alcoholic fatty liver disease (NAFLD) in humans and animals has been extensively studied (8)(9)(10), the hormone's short circulating half-life (30-120 min) and its tendency to aggregate in vitro limit its clinical application (11).To overcome these limitations, the long-acting FGF21 analog, PF-05231023, was developed.The analog possesses better anti-aggregation and in vivo degradation effects than that of natural FGF21 (12)(13)(14).PF-05231023 contains two modified human FGF21 molecules that are linked to the humanized immunoglobulin 1 antibody backbone, which was designed to prolong the analog's half-life and bioavailability (15).Compared with that of natural FGF21, PF-05231023 exhibits a 70-fold increase in half-life, demonstrating promising clinical potential in vitro.Similarly, administration of LY2405319, an FGF21 analog, reduced atherosclerotic plaques and blood lipid levels in Apoe −/− mice (16).Clinical trials have demonstrated the potential of recombinant FGF21 for the treatment of type 2 diabetes, obesity, and other comorbidities, but its physiological role in atherosclerosis (AS) has only gained attention in recent years (16,17).
However, the specific mechanism by which PF-05231023 modulates lipid metabolism and protects against AS in Apoe −/− mice remains unclear, and its impact on lipid metabolism-related pathways has yet to be elucidated.Therefore, we hypothesized that PF-05231023 would improve lipid metabolism and atherogenesis in Apoe −/− mice.Hence, in this study, we used an Apoe −/− mouse model of lipid metabolism disorder, using mice fed a Western diet equivalent (WD), to investigate the specific mechanism by which PF-05231023 alleviates lipid metabolism disorders.We investigated this from a novel perspective of adipose tissue, with the expectation of providing insights for the development of more effective and safer drugs for the treatment of metabolic diseases.

. Animal experiments
Eighteen 4-week-old female Apoe −/− C57BL/6 mice, weighing 17-20 g, were purchased from the Shanghai Model Organisms Center, Inc. (Shanghai, China).The animal quality was certified as SYXK (Shanghai, China) 2019-0002.Six 4-week-old C57BL/6 female mice with the same genetic background, weighing 16-17 g, were procured from the Guangdong Medical Experimental Animal Center with an animal quality license of SYXK (Guangdong, China) 2022-0002.All animal experiments strictly complied with the regulations of the Animal Ethics Committee of South China Agricultural University.The mice were housed and fed at room temperature (22 ± 2 • C), with a 12-h light/12-h dark cycle and had free access to food and drinking water.

. Experimental design
After acclimating to the environment for 1 week, the mice were randomly divided into two groups: Apoe −/− + WD + PF-05231023 (n = 6) and Apoe −/− + WD + saline group (n = 6).These groups were fed a WD for 12 weeks to build the AS model, followed by a 7-week treatment period of either 10 mg/kg of PF-05231023 (15) or an equivalent volume of saline intraperitoneally injected on Monday, Wednesday, and Friday every week.Additionally, C57BL/6 female mice + control diet (WT + Control) served as the healthy control group (n = 6), and Apoe −/− mice + control diet (Apoe −/− + Control) served as the transgenic control group (n = 6).The composition of the WD and control diet is shown in Table 1.
Throughout the feeding period, the weights of the mice were recorded weekly.Their food and water were also replaced weekly.Body composition analysis of the mice was conducted at weeks 12 and 19 of feeding, and an intraperitoneal glucose tolerance test (IPGTT) was performed during the 7th week of treatment.Blood samples were collected from the eyeballs and perfused with physiological saline to dissect the aorta.Subsequently, the mouse liver, subcutaneous fat, brown fat, and white fat around the ovaries were stored at −80 • C for subsequent analysis.Experimental design is shown in Figure 1.

. Body composition analysis
Body composition analysis was performed on mice at weeks 12 and 19.Before testing, place the mice between two paper cups, gently squeeze them, and perform stress induced bowel movements to minimize experimental errors.Subsequently, they were placed in a live animal body composition analyzer to record their body weight, body fat weight, lean meat weight, and their percentage.

. IPGTT
Mice were transferred to a clean cage, where they were fasted for 5 h, during which they had access to water.After the fasting period, their weight was measured, and the appropriate amount of glucose injection was calculated based on their weight.Fasting blood glucose levels were measured, and a glucose solution was injected into the abdominal cavity at a standard concentration of 2 mg/g (19).Blood glucose levels were monitored at 15, 30, 60, 90, and 120 min post-injection using a blood glucose meter.

. Organize sample collection
Blood collection from the eyeballs: After anesthetizing the mice with ether (20), the mice were fixed with one hand, and the eyeballs were removed with sterilized forceps; the blood was collected into 1.5 ml tubes, and left to stand for 4 h.The blood was centrifuged at 3,000 rpm for 15 min at 4 • C in a centrifuge (Eppendorf, Hamburg, Germany), and the supernatant was pipetted into 200 µl tubes and store it in a −80 • C freezer for further research.
After completion of blood sampling, the mice were executed by dislocation method, and the lower limbs and head were fixed on a foam plate to take white adipose tissue around the ovaries (sexual fat), subcutaneous fat, and brown fat of mice.One side was fixed in paraformaldehyde for subsequent embedding; the other side was placed in liquid, the other side was placed in liquid nitrogen and stored in the refrigerator at −80 • C.
Then, the thoracic cavity of the mice was cut open from the diaphragm upward to expose the heart, and a small opening was cut out of the right apical heart, and pre-cooled saline was injected from the left apical heart with a 1 ml syringe until the outflow from the opening of the right apical heart was clear and the color of the liver was gradually changed to lighter; the aorta of the mice was dissected under the body microscope, and the peri-arterial blood vessels and fat were stripped off, and the aortic arch was fixed in paraformaldehyde for subsequent staining analysis (21).

. Biochemical analysis of serum and liver
The collected blood samples were centrifuged at 3,000 rpm for 15 min at 4 • C to separate the serum.Liver tissue (30 mg) was homogenized with phosphate-buffered saline, and the supernatant was collected for further analysis.Serum and liver TC, and TG levels were measured according to the manufacturer's instructions provided with the reagent kit.
. Histological analysis of liver, aorta, and adipose tissue White fat surrounding the left hepatic lobule, aorta, and left ovary was fixed in 4% paraformaldehyde for 24 h.Subsequently, the liver and fat were embedded in paraffin sections (thickness = 5 µm) and subjected to HE staining.Lipid accumulation in the liver and adipocyte hypertrophy in the fat were observed and photographed under a 20× biological microscope (SOPTOP, EX20, Suzhou, China).The aorta was longitudinally placed on a black gel, and blood vessels were cut longitudinally under a stereomicroscope (Olympus Corporation, SZ2-ILST, Tokyo, Japan).Oil Red O staining was performed, and lipid deposition in the aorta was observed using a 4× microscope (SOPTOP, EX20).Image View software was used for image stitching, and analysis was performed using ImageJ1 software.

. Extraction of RNA from mouse adipose tissue
Three mice were randomly selected from the Apoe −/− + WD + PF-05231023 and Apoe −/− + WD + saline group (n = 6).The white fat tissue surrounding the ovaries was collected, and total RNA was extracted from this adipose tissue using TRIzol reagent.The purity of samples was assessed using a NanoPhotometer R Thermo Fisher spectrophotometer (Massachusetts, USA), whereas the integrity and concentration of the RNA samples were determined using the Agilent Technologies (CA, USA) 2100 RNA Nano 6000 Assay Kit. .

Library construction and sequencing
Following total RNA extraction, eukaryotic mRNA was enriched using oligo (dT) magnetic beads.A buffer was then added for mRNA fragmentation, and mRNA was used as a template to synthesize the complementary DNA (cDNA) first and second strands.The cDNA was purified, and the resulting purified doublestranded cDNA was subjected to end repair, addition of base A, and addition of sequencing adapters.Fragments were screened to recover ∼350 bp of cDNA, and PCR enrichment was performed to obtain a cDNA library.After passing the quality inspection using Qubit 3.0 and Agilent 2100, the constructed library was sequenced using a high-throughput sequencing platform (Agilent Technologies Inc, California, USA) with a PE150 sequencing strategy.The sequencing was conducted by Annoroad Gene Technology (Beijing, China). .

Di erential gene analysis and functional enrichment
Gene differential expression analysis was performed using DESeq2, with screening criteria for differential genes set at FoldChange ≥1.5, p-value ≤ 0.05, and padj ≤1.For Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of differentially expressed mRNA, we used the Database for Annotation, Visualization, and Integrated Discovery (https://david.ncifcrf.gov/tools.jsp).We used the microbiome websites (http://www.bioinformatics.com.cn and https://www.omicshare.com/tools/)for analysis and visualization. .

Real-time fluorescence quantitative reverse transcription PCR
To validate RNA sequencing (RNA-Seq) data, eight differentially expressed genes (DEG) related to metabolism were randomly selected for quantitative reverse transcription PCR (qRT-PCR) validation, with β-actin serving as an internal reference gene.Primer sequences and related information are provided in Table 2.The RNA sample used for qRT-PCR amplification was the same as the RNA sample used for constructing the RNA-Seq library mentioned above when using the Vazyme reverse transcription kit. .

Statistical analysis
Unless otherwise specified, statistical analysis was conducted using one-way analysis of variance (ANOVA).We performed multiple comparisons after one-way ANOVA, compared each column, and rated the average of each column.Data is expressed as mean ± standard error mean, and p < 0.05 is considered statistically significant.All statistical analyses were performed using GraphPad Prism 8.0.

Results
. The e ects of PFadministration on body weight and fat in Apoe -/-mice During the first week of feeding, there was no significant difference in body weights among all mice, although the Apoe −/− mice exhibited a slightly higher body weight than that of the WT + Control group.After 12 weeks of the feeding regimen, there was no significant difference in weights between the Apoe −/− + WD + PF-05231023 group and the Apoe −/− + WD + saline group.However, a significant difference in weights was noted between the WT + Control group and the Apoe −/− + Control group (p < 0.05).PF-05231023 treatment was initiated after the 12th week.During the 7-week treatment period, the weight of mice in the Apoe −/− + WD + PF-05231023 group increased in the first week, gradually decreased, and then remained stable thereafter.The weight of the Apoe −/− + WD + saline group showed a slight upward trend; however, the difference was not significant between the two groups.Furthermore, a significant difference in body weight was noted between the Apoe −/− + Control group and the Apoe −/− + WD + saline group (p < 0.01).A significant difference in body weight was also observed between the WT + Control group and the Apoe −/− + Control group (p < 0.05, Figure 2A), as well as between the Apoe −/− + WD + PF-05231023 group and the Apoe −/− + WD + saline group before mouse sampling (p < 0.05, Figure 2B).These results indicate that PF-05231023 treatment affected the body weight of Apoe −/− mice.
Body composition analysis conducted before and after treatment revealed no significant fluctuation in fat content between the WT + Control and Apoe −/− + Control groups (Figure 2C).Both the Apoe −/− + WD + PF-05231023 and Apoe −/− + WD + saline groups showed a decrease in fat content before and

Gene
Accession number Frontiers in Veterinary Science frontiersin.orgafter treatment (Figure 2C).Specifically, the saline group showed a decrease of 2.99%, whereas the PF-05231023 group exhibited a decrease of 6.82%.The proportion of decrease in the PF-05231023 group was more than twice that in the control group.Overall, the proportion of fat that decreased in the PF-05231023 group was higher than that in the saline group (Figure 2D).These results indicate that PF-05231023 causes s a slight decrease in fat content in Apoe −/− mice.
. The e ects of PF-on blood glucose levels in Apoe −/− mice At the end of 7 weeks of treatment, mice were subjected to an IPGTT.Within 120 min of the IPGTT, mice treated with PF-05231023 showed the fastest decrease in blood glucose levels at 30 min compared with that in the group injected with physiological saline (Figure 3A).The area under the glucose curve was also lower than that for the control group (Figure 3B).Although blood glucose levels were slightly higher in the fasting state (Figure 3C), no statistical difference was observed.These results indicate that the effect of PF-05231023 treatment on blood glucose levels in Apoe −/− mice was modest.Plasma lipid level analysis revealed no significant decrease in TC and TG levels in the plasma of mice treated with PF-05231023 compared with that of mice treated with physiological saline (data not shown).
. The e ects of PF-on the aorta of Apoe −/− mice Oil Red O staining analysis was performed on the aortic arch.The WT + Control group exhibited a normal aortic wall with no lipid deposition.However, partial lipid deposition was observed in  µm.*means there is a significant di erence between the two groups (p < .), **means there is a highly significant di erence between the two groups (p < .).
the aortic wall of the Apoe −/− + Control group.The intima of the aorta in the Apoe −/− + WD + saline group appeared relatively fragile, with a substantial amount of lipid deposition noted on the vessel wall.Compared with that in the saline injection group, the Apoe −/− + WD + PF-05231023 group showed reduced lipid deposition on the vessel wall and milder plaque lesions (Figure 4A).Furthermore, the plaque area was significantly smaller compared with that in the saline injection group (Figure 4B; p < 0.05).These .The e ects of PF-on lipid deposition and biochemical levels in the liver of Apoe −/− mice Liver sections stained with HE were examined under a microscope at 200× magnification.Compared with that in the group injected with saline, mice treated with PF-05231023 exhibited a smaller area of hepatic fat vacuolization (Figure 5A) and a significant decrease in lipid deposition (Figure 5B, p < 0.01).This decrease approached the level observed in the Apoe −/− + Control group, indicating that PF-05231023 improved hepatic steatosis in Apoe −/− mice.In addition, the TC levels in the livers of mice from the Apoe −/− + WD + PF-05231023 group were lower than those in the saline group (Figure 5D).Although TG levels did not show statistical differences, there was a slight trend of decrease (Figure 5E).PF-05231023 also improved liver weight (Figure 5C).
. The e ects of PF-on Apoe −/− mouse adipose tissue Sections of white adipose tissue around the ovaries were subjected to HE staining and examined using a microscope at 200× magnification.The adipocytes in the WT + Control group mice were tightly arranged, had small cell diameters, and possessed small volumes (Figure 6A).Compared with those in the WT + Control group, the Apoe −/− + Control group exhibited slightly larger cell diameters and volumes (Figure 6A).The volume and diameter of adipocytes in the Apoe −/− + WD + saline group were increased, exhibiting characteristics of uneven cell size and irregular shape (Figure 6A).However, in the Apoe −/− + WD + PF-05231023 group, adipocytes were smaller than those in the Apoe −/− µm.*means there is a significant di erence between the two groups (p < .), **means there is a highly significant di erence between the two groups (p < .).

Frontiers in Veterinary Science
frontiersin.org*means there is a significant di erence between the two groups (p < .), **means there is a highly significant di erence between the two groups (p < .).
+ WD + saline group, with reduced volume and diameter and more regular arrangement (Figure 6A).The average area of white adipocytes around the ovaries significantly decreased in the average area of adipose tissue cells in the Apoe −/− + WD + PF-05231023 group compared with that in the Apoe −/− + WD + saline group (Figure 6B, p < 0. 05).Weight statistics were obtained for white, subcutaneous, and brown fat around the ovaries after sampling.Compared with that in the Apoe −/− + WD + saline group, the Apoe −/− + WD + PF-05231023 group showed a significant decrease in the weight of white fat around the ovaries (Figure 6C) and subcutaneous fat (Figure 6D, p < 0.05), whereas no statistically significant difference was noted in terms of brown fat (Figure 6E, p > 0.05).

. DEGs and functional annotation analysis
We conducted sequencing analysis of the white adipose tissue around the ovaries in the Apoe −/− + WD + PF-05231023 group and the Apoe −/− + WD + saline group.Three biological replicates were used for each group.Our sequencing results revealed a total of 3,749 DEGs between Apoe −/− + WD + PF-05231023 and Apoe −/− + WD + saline mice, including 1,538 upregulated genes and 2,211 downregulated genes (Figure 7A; screening parameter | log 2 Fold change | ≥ 1 and q < 0.05).The volcano plot analysis a more intuitive up-and-down regulation of DEGs (Figure 7B).Principal component analysis of these DEGs demonstrated high similarity within the groups (Figure 7C).We used the GO and KEGG databases to classify DEG functions (Figure 8).In the top 10 biological processes (BP), cellular component (CC), and molecular function (MF), GO enrichment analysis showed that BP was primarily associated with translation, aerobic respiration, lipid metabolism, and immune response processes, whereas CC was mainly concentrated in mitochondria, ribosomes, respiratory chains, and other cellular parts.In MF, enrichment was observed in ribosomal structural components, oxidoreductase activity, and homologous protein binding, and other functional aspects (Figure 8A).We also annotated DEGs based on the KEGG database and noted that 3,749 DEGs were concentrated in 81 KEGG pathways within six categories: metabolism, genetic information processing, environmental information processing, cellular processes, biological systems, and human diseases.Among these pathways, apart from human diseases, the most DEGs were in metabolic processes, including oxidative phosphorylation, metabolic pathways, fatty acid metabolism, steroid biosynthesis, glutathione metabolism, and propionic acid metabolism (Figure 8B).
Six pathways were closely related to lipid metabolism in the KEGG analysis, including fatty acid metabolism, steroid biosynthesis, fatty acid elongation, fatty acid biosynthesis, fatty acid degradation, and cholesterol metabolism, as shown in Figure 9.In the fatty acid metabolism pathway, key genes involved in fatty acid synthesis, such as fatty acid synthase (Fasn), acetyl CoA carboxylase alpha (Acaca), and acetyl CoA carboxylase beta (Acacb), were significantly downregulated, whereas key genes involved in fatty acid oxidation, such as Cpt1ab, were upregulated.In the fatty acid elongation pathway, Mecr, very long chain fatty acid elongase 1 (Elovl1), and very long chain fatty acid elongase 6 (Elovl6) were significantly downregulated.In the steroid biosynthesis pathway, genes related to cholesterol synthesis, such as farnesyl-diphosphate farnesyltransferase 1 and squalene epoxidase, were significantly downregulated.In addition, steroyl CoA depletion 1 (Scd1) and adipogenin, which are involved in fat production, were significantly downregulated, whereas adenosine triphosphate-binding cassette transporter G8 (Abcg8), which promotes cholesterol efflux, was significantly upregulated.The levels of sterol regulatory element binding transcription factor 1 (Srebf1) also significantly decreased.Table 3 provides a detailed description of the genes involved in these pathways.

. Real-time fluorescence quantitative PCR validation results
To verify the reliability of the transcriptional data, we randomly selected nine DEGs with high expression levels for quantitative PCR (qPCR) analysis.The qPCR results were consistent with the trends observed in the transcriptome analysis, confirming the accuracy and reliability of the transcriptome sequencing results (Figure 10A).Moreover, a high correlation was observed between the qPCR and RNA-Seq data, with R 2 = 0.943 (Figure 10B).

Discussion
FGF21 analogs have been studied in clinical trials for type 2 diabetes and NAFLD and have demonstrated generally good tolerability.This study confirms that the long-acting FGF21 analog PF-05231023 reduces the accumulation of atherosclerotic plaques and improves lipid metabolism in the liver and adipose tissue of Apoe −/− mice, thereby indicating the clinical potential of the FGF21 analog in treating AS.In this study, the body weights of Apoe −/− mice treated with PF-05231023 gradually decreased until changes were observed prior to sampling.This result aligns with those of previous studies that reported weight loss in response to administration of FGF21 analogs in diet-induced obese mice (DIO) (22), and in Gubra Amylin NASH DIO mice (10).Although some clinical trials have suggested weight reduction after FGF21 analog treatment in subjects with obesity, type 2 diabetes, and fatty liver disease, the evidence for weight loss in subjects with obesity remains inconclusive (23).For example, in a clinical trial evaluating the safety and tolerability of PF-05231023 in patients with obesity with hypertriglyceridemia, PF-05231023 reduced TG and lowdensity lipoprotein cholesterol levels without causing body weight reduction (24).After 7 weeks of treatment, the WT + Control and Apoe −/− + Control groups showed minimal changes in body fat percentage.Conversely, both the PF-05231023 treatment and the physiological saline control groups showed a declining trend in body fat percentage, with the PF-05231023 treatment group demonstrating a more significant decrease.We hypothesize that this reduction in body fat percentage is likely attributed to the weekly intraperitoneal injections administered to both mouse groups, causing a certain degree of stress.This is supported by the observation that the two control groups, which did not receive intraperitoneal injections, did not show significant changes in body fat.FGF21 reduces blood sugar levels in diabetic rodents, obese mice, and monkey models (10).However, this study found that although there was no statistically significant difference in fasting blood glucose levels between Apoe −/− mice injected with PF-05231023 and those injected with physiological saline, the former group showed an improvement in glucose tolerance.PF-05231023 does not significantly affect blood glucose control (11,15).These minor differences in glucose improvement may be attributed to the slight differences in the pharmacology of FGF21 analogs (15).Additionally, injection of PF-05231023 had no significant effect on the reduction of plasma lipids in Apoe −/− mice.Similarly, no significant reduction in plasma TG levels was detected in DIO mice treated with FGF21 (25).A study conducted on obese Göttingen mini pigs also found that FGF21 had no effect on pig plasma lipids, partly because of the very low circulating lipid levels in the mini pigs, which rendered FGF21 ineffective in reducing pig plasma lipids (26).The reason plasma lipid levels exhibited no significant effect in the current study remains unclear and could be a topic for further research.Numerous studies have investigated the role of FGF21 in AS.For example, a study using Apoe −/− mice demonstrated that FGF21 alleviated AS by enhancing Fas-mediated apoptosis (27).Another study reported that FGF21 upregulates autophagy-mediated cholesterol efflux via receptor for activated C kinase 1 to inhibit AS (17).Furthermore, FGF21 alleviates AS by inhibiting focal pyroptosis in vascular endothelial cells, mediated by the NLR family pyrin domain containing three inflammasome (28).Thus, several mechanisms have been proposed for the inhibition of AS.In our study, PF-05231023 administration reduced fat accumulation in the liver and significantly reduced TC and TG content in the liver.This result aligns with the conclusion that PF-05231023 treatment reduces liver TC and TG in NAFLD, and reduces plasma liver injury markers alanine aminotransferase and aspartate aminotransferase (10).Additionally, our results are also consistent with research indicating that FGF21 improved lipid droplet size in the liver (16), as did the FGF21 analog LY2405319 (12).In various dietinduced, genetically modified, or chemically induced models, PF-05231023 treatment reduces the weight of brown fat, subcutaneous fat, and white adipose tissue, and it reduces adipocyte size in the white adipose tissue around the ovaries.FGF21-specificknockout mice exhibit weight gain, decreased lipolysis, and reduced energy expenditure in the adipose tissue, leading to adipose tissue hypertrophy (11).However, FGF21 treatment reduces weight gain, improves lipid levels in adipocytes, and reduces inflammatory cell infiltration (29).To better understand the molecular pathways associated with PF-05231023 and its effect on lipid metabolism in adipose tissue, we analyzed gene expression in the white adipose tissue around the ovaries.First, we annotated DEGs using the KEGG database and noted that most of the DEGs were concentrated in the categories related to metabolism and human diseases.By combining KEGG and GO functional analyses, we observed that pathways related to metabolism and human disease were downregulated in the differential gene set.Enrichment results for human disease show that PF-05231023 has a significant positive impact on the pathways associated with neurodegenerative diseases, including Alzheimer's disease (30), Parkinson's disease (31,32), Huntington's disease, and amyotrophic lateral sclerosis.This finding suggests a potential role for longacting FGF21 analogs in neuroprotection.FGF21 can modulates anti-inflammatory and antioxidant stress responses through nuclear factor kappa-B and adenosine 5 ′ -monophosphate-activated protein kinase/protein kinase B signaling pathways, thereby protecting nerves from damage and mitigating neurodegenerative changes (33).Furthermore, our analysis revealed that PF-05231023 significantly downregulated the expression of genes related to NAFLD.This downregulation would lead to an improvement in the symptoms of this disease (34).This beneficial effect of PF-05231023 may be attributed to the direct regulation of lipid metabolism through insulin, resulting in reduced lipid accumulation in the liver.In the metabolic category, we identified several DEGs involved in metabolic pathways and lipid metabolism.We found that PF-05231023 significantly downregulated Srebf1, further leading to a decrease in the expression of Fasn, Acaca, Acacb, and Scd1.Srebf1 is an important transcription factor that regulates lipid metabolism related enzymes primarily expressed in the liver and adipocytes.The transcription factor induces de novo adipogenesis by regulating the expression of adipogenic genes, such as Acaca and Acacb, Fasn (35), and Scd1 (36, 37).Acaca and Acacb catalyze rate-limiting reactions in the biogenesis of long-chain fatty acids (38).Fasn is a key synthase in the process of fatty acid biosynthesis (38), which catalyzes de novo fatty acid synthesis by converting acetyl CoA and malonyl CoA to palmitic acid esters (39), and Scd1 regulates the expression of Frontiers in Veterinary Science frontiersin.orgunsaturated fatty acid biosynthesis genes and mitochondrial fatty acid oxidation (38).Moreover PF-05231023 also reduced the expression of adenosine triphosphate citrate lyase, which is an important enzyme in the cholesterol and fatty acid biosynthesis pathway upstream of Fasn (38), thereby promoting the biosynthesis of cholesterol and fatty acids by producing acetyl CoA from citrate (40).Elovl1 and Elovl6 are downregulated by Srebf1, which also leads to a decrease in fatty acid synthesis.Moreover, we observed a significant upregulation of Abcg8 and an upregulation trend of adenosine triphosphate-binding cassette transporter G5 (Abcg5).The ABCG5/ABCG8 complex transports cholesterol and phytosterols from intestinal cells to the intestinal cavity for fecal treatment, thereby promoting cholesterol efflux and reducing cholesterol accumulation (41).

Conclusion
In summary, we confirmed that PF-05231023 reduces liver cholesterol and TG levels as well as lipid deposition in WD-fed Apoe −/− mice and has the potential to counteract the development of AS.Further analysis of adipose tissue indicated that PF-05231023 reduces the expression of genes involved in adipogenesis, cholesterol synthesis, and fatty acid derivative pathways.Moreover, we found that the specific mechanism by which PF-05231023 inflicts these effects is to inhibit the expression of Srebf1 and further inhibit the expression of genes related to lipid generation, thereby reducing lipid synthesis.However, the specific role of this pathway requires further investigation.

. Study limitations
In the current study, we revealed the ameliorative effects of PF-05231023 on lipid metabolism as well as inhibitory effects on the expression of lipogenesis-related genes.However, the specific pathways regulated by PF-05231023 remain to be fully elucidated.Hence, the following aspects need to be further investigated: the mechanism by which PF-05231023 down-regulates Srebf1 expression, whether Srebf1 is linked to the expression of other lipogenesis-related genes, and the regulatory pathways between Srebf1 and other lipogenesisrelated genes.

FIGURE( A )
FIGURE (A) Changes in body weight of Apoe −/− mice during feeding and treatment.*indicates a significant di erence between the WT + Control group and the Apoe −/− + Control group (*p < ., **p < .), whereas # indicates a significant di erence between the Apoe −/− + Control group and the Apoe −/− + WD + saline group ( # p < ., ## p < .).Control represents the Western-style diet control group, and WD indicates the Western-style diet purification group.(B) Body weight of mice before sampling.(C) Fat content before and after treatment.(D) Fat mass change after treatment.

FIGURE(
FIGURE (A) Detection of glucose levels within -min of the intraperitoneal glucose tolerance test (IPGTT) in mice at the end of treatment, *indicates a significant di erence between the WT + Control group and the Apoe −/− + Control group (B) Increment of area under the curve (iAUC) of IPGTT across the four groups.(C) Fasting blood glucose concentration in di erent groups of mice after weeks of treatment.GC, glucose.

FIGURE( A )
FIGURE (A) Representative images of Oil Red O-stained aortic arch from the four mouse groups.Red stain indicates plaque accumulation.(B) Atherosclerotic plaque area after treatment.Scale bar:µm.*means there is a significant di erence between the two groups (p < .), **means there is a highly significant di erence between the two groups (p < .).
PF-05231023 reduced plaque accumulation in the aortic arch of Apoe −/− mice.

FIGURE(
FIGURE (A) Representative images of hematoxylin and eosin (HE)-stained livers from the four mouse groups.The black arrows indicates the main lesions.(B) Percentage of liver vacuolization.(C) Liver weight after sampling.(D) Total cholesterol (TC) content in the liver.(E) Liver triglyceride (TG) levels.Scale bar:µm.*means there is a significant di erence between the two groups (p < .), **means there is a highly significant di erence between the two groups (p < .).

FIGURE( A )
FIGURE (A) Representative images of Hematoxylin and eosin (HE)-stained white adipose tissue around the ovaries of mice in the four study groups.(B) Average area of white adipocytes around the ovaries.(C) Weight of white fat around the ovaries after sampling.(D) Subcutaneous fat weight after sampling.(E) Weight of brown fat after sampling.Scale bar:µm.*means there is a significant di erence between the two groups (p < .), **means there is a highly significant di erence between the two groups (p < .).

FIGURE(
FIGURE(A) Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of genes related to lipid metabolism and some significantly di erent genes.(B) Heat map depicting changes in candidate gene expression related to lipid metabolism.Blue indicates significant downregulation of gene expression (p < .), whereas red indicates significant upregulation of genes (p < .).

FIGURE
FIGUREValidation of the selected di erentially expressed genes (DEG) using quantitative reverse transcription PCR (qRT-PCR).(A) Comparison of expression trends between the RNA sequencing (RNA-Seq) and qRT-PCR data.(B) Correlation between selected gene expression ratios measured using RNA sequencing and qRT-PCR.
TABLE Western diet equivalent (WD) and control diet compositions.
TABLE DEGs involved in de novo adipogenesis, cholesterol biosynthesis, and fatty acid elongation pathways.